1. Field of the Invention
The instant invention is drawn to a method for the structure-based design of inhibitors of DNA polymerase, and DNA repair enzymes, to methods for inhibiting DNA replication and repair, and to methods for treating viral infections, bacterial infections, fungal infections, protozoan infections, and neoplastic diseases.
2. Description of the Related Art
The herpesviruses, herpes simplex virus (HSV), and human cytomegalovirus (CMV), are two important human pathogens. HSV causes a spectrum of diseases in immunocompetent adults including debilitating genital infections, sight-threatening ocular infections and occasionally encephalitis that is also debilitating and can be fatal if untreated (Corey, L., and P. C. Spear. 1986 N. Engl. J. Med. 314, 686-691.). In newborns and immunosuppressed individuals such as AIDS patients, HSV infections are even more severe. CMV causes little disease in immunocompetent adults, but it is a major cause of birth defects and a major pathogen in immunosuppressed individuals, especially AIDS and transplant patients (Britt, W. J., and C. A. Alford. 1996. Cytomegalovirus, 3rd Edition ed. In Fields Virology. B. N. Fields, D. M. Knipe, P. M. Hawley, R. M. Chanock, J. L. Melnick, T. P. Monath, B. Roizman and S. E. Straus, editors. Lippincott-Raven, Philadelphia. 2493-2523). There is also evidence for a role of CMV in cardiovascular diseases (e.g., Zhou et al. 1996 N. Engl. J. Med. 335, 624-630.).
Ideally, a target for an antiviral drug should be a viral gene product that differs significantly from host functions and is either essential for viral replication or can activate a drug that inhibits viral replication. Most antiherpesvirus drugs developed to date have targeted herpesvirus thymidine kinases (TK) to activate drugs, and herpesvirus DNA polymerases to be inhibited by the drugs (Coen, D. M. 1992. Sem. Virol. 3, 3-12). For example, HSV TK, which is not essential for replication in cell culture, activates acyclovir (ACV) by phosphorylation to its monophosphate much more efficiently than do cellular enzymes. Cellular enzymes convert the monophosphate to the triphosphate, which is a selective and potent inhibitor of HSV DNA polymerase (Pol), which is essential for replication. That TK and Pol serve as selective drug targets has been established both by biochemical studies and by the isolation and analyses of drug resistant mutants (Coen, D. M. 1986 J. Antimicrob. Chemother. 18, 1-10).
However, nearly all drug-resistance Pol mutations map in regions encoding regions of Pol that are conserved with human cellular DNA polymerases (Coen, D. M. 1996. In Antiviral Drug Resistance. D. Richrnan, editor. John Wiley & Sons, Chichester. 81-102). Thus, these antiviral drugs appear to exploit only rather subtle differences between viral and cellular and polymerases. Moreover, there are HSV infections for which these drugs are not particularly efficacious and there remain concerns about the potential for toxic effects over the lifetime of a patient and the increasing number of cases in which resistance to these drugs develops (Safrin, S. 1996. In Antiviral Drug Resistance. D. D. Richman, editor. John Wiley & Sons, Chichester. 103-122).
In an alternative investigative approach, other polymerase sites are targeted for inhibition. Within this approach, it has been observed that herpesviruses require a specific interaction between HSV DNA polymerase and a processivity factor to effect synthesis of long strands of DNA. In the case of HSV, the accessory proteins, UL42, functions by increasing processivity (Gottlieb et al., 1990 J. Virol. 64, 5976-5987). Both Pol and UL42 are essential for virus replication and this essentiality extends to the analogous proteins encoded by other herpesviruses that have been examined. Mutations that specifically disrupt HSV Pol-UL42 interactions block long chain DNA synthesis and viral replication indicating that these interactions are essential for virus replication (Digard et al., 1993 J. Virol. 67, 398-406; Digard et al., 1993 J. Virol. 67, 1159-1168). The segment of Pol that interacts with UL42 has been mapped to the C-terminus of the enzyme by a combination of genetic and biochemical methods (Digard et al., 1993 J. Virol. 67, 398-406; Digard P., and Coen, D. M. 1990 J. Biol. Chem. 265, 17393-17396; Marsden et al., 1994 J. Gen. Virol. 75(Pt 11), 3127-3135.; Stow et al., 1993 Nucleic Acids Res. 21(1), 87-92.; Tenney et al., 1993 J. Virol. 67(1), 543-547.) Peptides corresponding to the C-terminal segment of Pol specifically block long chain DNA synthesis by Pol-UL42 in vitro (Digard et al., 1995 Proc. Natl. Acad. Sci. 92, 1456-1460; Marsden et al., 1994 J. Gen. Virol. 75(Pt 11), 3127-3135.) and interfere with HSV infectivity in tissue culture (Loregian et al., 1999, Proc. Natl. Acad. Sci. 96: 5221-5226). This segment of Pol is partially helical (Digard et al., 1995 Proc. Natl. Acad. Sci. 92, 1456-1460.) but there has been no information about the structure of UL42.
The best understood processivity factors are known as sliding clamps, which include the Escherichia coli β-subunit of DNA polymerase III, bacteriophage T4 and RB69 gp45, and the eukaryotic clamp, PCNA. These proteins do not bind directly to DNA but, rather, form multimeric rings around DNA, which permits them to slide along the template. Moreover, under physiological conditions, the association of a sliding clamp with DNA and its cognate polymerase requires auxiliary proteins that serve as “clamp loaders’ (Kuriyan, J., and O'Donnell, M. 1993 J. Mol. Biol. 234, 915-925.). UL42 differs from sliding clamps in that it binds directly and stably to DNA and does not require additional factors to load onto Pol or DNA (Gottlieb, J., and Challberg, M. D. 1994 J. Virol 68, 49374945; Marsden et al., 1987 J. Virol. 61, 2428-2437; Powell, K. L., and Purifoy, D. J. M. 1976 Iniervirol. 7, 225-239; Weisshart et al., 1999 J. Virol. 73, 55-66). There have been two reports of structures of processivity factors bound to peptides: 1) human PCNA complexed with a 22 residue peptide derived from the C-terminus of the cell cycle checkpoint protein p21 (Gulbis et al., 1996 Cell 87, 297-306); and 2) gp45 from the phage RB69 complexed with a C-terminal peptide fragment of the RB69 DNA polymerase (protein data base 1B77 1B8H; Shamoo et al., 1999, Cell, 99:155-166).
Accordingly, the interaction between processivity subunits and proteins whose functions depend upon processivity factor binding, may be an especially amenable drug target relative to other protein-protein interactions. However, many protein-protein interactions involve large surfaces which involve multiple binding site interactions. Accordingly, an effective method by which structure-based design of molecules inhibiting binding between proteins and a processivity factor subunit is desired. Moreover, a method for treating infections (i.e., viral, bacterial, fungal) and methods for treating cancer and tumor growth with structure-based design inhibitors of processivity factor binding are particularly desired.